Deformed wing virus (DWV) is a serious pathogen of the honeybee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In present study a novel micro PCR-based detection method, termed as ultra-rapid real-time PCR (UR-RT PCR), was developed for the fast and quantitative detection of DWV in honeybee. A specific detection primer set (DWV-UR-F3/R3) was used for the amplification of an unique 133-bp DNA fragment of DWV with a rapid real -time PCR system, GenSpector® TMC-1000, which proceed the cycling with fast heating and cooling rates and a small reaction volume. We showed that this method is able to detect DWV with DNA conditions, artificial recombinant DNA, pBX-DWV479 as well as with virus-infected honeybee samples. In application to a DWV-infected honey bee, the minimum detection time was 8 min 50 seconds under 30 cycles and 10min 11 seconds including melting temperature analysis. This optimizing detection method is one of the fastest real-time PCR-based diagnostic tools and is available to be applied to use for the detection in the field and of various persistency pathogens.

Sacbood virus (SBV) is an infectious disease, resulting in failure to pupate and death and kSBV is a disease caused perish Apis cerana of 75% in South Korea. RNA dependent RNA Polymerase(RdRP) is one of polyprotein of viral genome and an enzyme that catalyzes the replication of RNA from an RNA templates and an essential protein encoded in the genomes of all RNA containing viruses with no DNA stages. In this study, recombinant construct with RdRP partial region of kSBV was used for sequence analysis to clarify about Korean SBV. As a result it could be determined that the virus develops by infection of Korean Apis cerana called kSBV. Also, we named Apis cerana-kSBV-region to the name of the unique region of gene that kSBV has. In comparison of the RdRP region of bee RNA virus on nucleotide sequence, its sequence from same species have less variability as well as each virus species has a certainty of RdRp region. It indicated that mutations of RdRP region of each virus species is able to be a useful indicator of honeybee virus detection.

Deformed wing virus(DWV) is one of infectious disease of honey bee that is caused to wings of immature or mutation and death at last. In this study 4 kinds of polyprotein of DWV are compared and then selected 2 kinds of polyprotein, RNA-dependent RNA polymerase(RdRP) and peptidase C3G, which has relatively higher homology than others. Analysis of both RdRP region and peptidase C3G of DWV deposited in Genbank of NCBI showed 99~100% and 95~97% of homology on genomic level, respectively whereas analysis of CRPV-capsid and RNA helicase showed 86~98% and 89~98% of homology, repectively. According to the result of gene analysis, two kinds of polyprotein are constructed and analyzed the homology, resulting in RdRP and Peptidase -C3G showed about 96% and 97% of homology, respectively. It indicated that both region is able to use for generation of specific antibody for the diagnosis of Deformed Wing Virus (DWV).

The infectious pathogens against honeybee (Apis mellifera) comprise a heterogeneous group of bacterial, viral, and fungal organisms including Paenibacillus larvae, Deformed Wing Virus (DWV) and Nosema apis. Many species like Paenibacillus larvae, Deformed Wing Virus (DWV) and Nosema apis have been isolated from a number of different continents, e.g. America, Asia and Europe, indicating its wide spread in whole nature. Little is known about the occurrence and distribution in the environment of these pathogens. For a more rapid, systematic and efficient monitoring of each pathogenic species against honeybee in the environment, PCR-based detection systems were developed that allows species-specific identifications of various pathogenic species with one reaction. These could be achieved by selecting specific primers from conserved regions of each species with speciesspecific DNA sequence variations. For the detection of any already known pathogen, well-developed PCR-detection system allows the specific detection of expected pathogenic species based on its specific nucleic acid sequence. Since each pathogenic species delivers a specific PCR-product of different size, bands can be distinguished very easily by simple gel electrophoresis. After the development of real-time PCR system, PCR-based specific detections of honeybee pathogens were dramatically improved their applications, from just detection to quantification of pathogens. These systems, quantitative PCR (qPCR) for the detection of honeybee pathogens, could be distinguished from previous PCR detection on the points of “real-time”, “easy” and “quantitative”. Moreover, very rapid PCR, so-called “Ultra-Rapid Real-Time PCR” were developed recently in field of pathogen-detection. Typical Honeybee pathogens such as Paenibacillus larvae, Israelli acute paralysis virus (IAPV) were successfully detected inner 7 minutes using 30 cycled Ultrarapid PCR. According to development of more rapid apparatus, even 30 cycled, 1 minute PCR seems to be possible. Ultla-Rapid PCR was currently attempted to apply for the direct detection system of all viral pathogens against honeybee from bee-samples and different environmental probes.